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Background Multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini. Methods We investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009–2010; (2) MDR strains from the Nhlangano region, 2014–2017). Results MDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987–1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2. Conclusion The high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.

Data presented in this study is publicly available at https://figshare.com/s/45bd5369a97b4c2489bf (accessed on 25 October 2021).Ethical permission for the study was provided by the University of Pretoria (ECO20-14), and this manuscript has been approved for publication by the Royal Veterinary College (PPS_01715). Permission to carry out research in the region was granted by the Northern Cape Department of Environment and Nature Conservation.; International audience; Simple SummaryDiagnostic tests used in animals are ideally extensively trialled to ensure that practitioners have confidence in the results; the ideal target should be 100% of infected animals testing positive (sensitivity), and 100% of uninfected animals testing negative (specificity). In these trials, a gold standard is necessary, against which the diagnostic tests may be compared. Commonly, tests of wild animals are not extensively trialled due to shortages of samples and the rarity of gold standard tests. This study uses samples collected for the purpose of diagnosing Mycobacterium suricattae infection in meerkats and estimates the sensitivity and specificity of available tests, both individually, and in combination. A small number of post-mortems (gold standard) were carried out, providing definitive evidence of infected animals against which to compare the tests. In addition, test results were unconventionally compared to survival times and clinical characteristics, aiming to quantify the prognostic capabilities of the tests. The study therefore not only estimates the required parameters against which to assess these tests, but also provides a model for future assessment of diagnostic tests in imperfect field scenarios. Wildlife diseases are increasingly recognised as important to society, and so methods to better quantify these are increasingly important.AbstractDiagnostic tests are used to classify individual animals’ infection statuses. However, validating test performance in wild animals without gold standard tests is extremely challenging, and the issue is further complicated in chronic conditions where measured immune parameters vary over time. Here, we demonstrate the value of combining evidence from different diagnostic approaches to aid interpretation in the absence of gold standards, large sample sizes, and controlled environments. Over a two-year period, we sampled 268 free-living meerkats (Suricata suricatta) longitudinally for Mycobacterium suricattae (a causative agent of tuberculosis), using three ante-mortem diagnostic tests based on mycobacterial culture, and antigen-specific humoral and cell-mediated immune responses, interpreting results both independently and in combination. Post-mortem cultures confirmed M. suricattae infection in 22 animals, which had prior ante-mortem information, 59% (13/22) of which were test-positive on a parallel test interpretation (PTI) of the three ante-mortem diagnostic assays (95% confidence interval: 37–79%). A similar ability to detect infection, 65.7% (95% credible interval: 42.7–84.7%), was estimated using a Bayesian approach to examine PTI. Strong evidence was found for a near doubling of the hazard of death (Hazard Ratio 1.75, CI: 1.14–2.67, p = 0.01), associated with a positive PTI result, thus demonstrating that these test results are related to disease outcomes. For individual tests, small sample sizes led to wide confidence intervals, but replication of conclusions, using different methods, increased our confidence in these results. This study demonstrates that combining multiple methodologies to evaluate diagnostic tests in free-ranging wildlife populations can be a useful approach for exploiting such valuable datasets.